1. Field of the Invention
The present invention is directed to glycosylated antibodies and antibody fragments modified to have reactive ketone groups at specific sites. These “landscaped” antibodies and antibody fragments can be conjugated with linkers, peptides, oligosaccharides or other agents useful in clinical applications having a ketone-reactive group, and used to deliver the agents to in vivo target sites.
2. Description of Related Art
Antibody immunoconjugates are widely used in modern medicine. Chemical methods allowing effective conjugation of a variety of diagnostic and therapeutic compounds, including drugs and chelates, to monoclonal antibodies (mabs) are well documented. However, most of these methods rely on random attachments to certain amino acid residues, such as tyrosine, lysine, aspartic acid and glutamic acid. BR-96-DOX 16771 and LL2-pseudomonas exotoxin immunoconjugates, which have demonstrated significant anti-tumor activity in tumor-bearing mice, are examples of antibody immunoconjugates constructed through conjugations at these residues. However, because these conjugates are made under extreme chemical conditions (non-physiological pHs, temperature, solvents, etc.) and because the conjugation is not site-specific, the resulting immunoconjugates may exhibit reduced and heterogenous antigen binding properties.
Rodwell et al., Proc. Nat'l Acad. Sci., 83: 2632 (1986), reported site-specific covalent modification of monoclonal antibodies (mAbs) using the Asn-linked carbohydrate (CHO) in the CH2 domain (Asn297) as a convenient chemical handle for radionuclide conjugation. 131I-conjugates formed by this method exhibited homogenous binding properties with improved in vivo targeting efficiency in mice. By using a soluble amino-dextran as an intermediate carrier, therapeutic drugs, such as methotrexate (MTX), flourouridine, or doxorubicin (DOX), have been conjugated at the CH2-appended carbohydrate moiety. See, for example, U.S. Pat. No. 4,699,784. However, because the Asn297-associated CHO is positioned at the internal space formed between the two adjacent CH2 domains, steric hindrance is expected to impede the efficiency of conjugation at this site. Moreover, antibody fragments, such as F(ab′)2, Fab′ and Fab, which often are preferred for clinical use, lack the Fc portion and the associated carbohydrate moiety. Accordingly, these species can not be conjugated by this method.
Hansen et al., U.S. Pat. No. 5,443,953, and Leung et al., U.S. Provisional Patent Application 60/013,709, the entire contents of which are incorporated herein by reference, describe the introduction of multiple glycosylation sites on the Vκ and CH1 (HCN1 and HCN5 sites) domains of antibodies. Attachment of chelates at all of these sites does not affect the immunoreactivity of the resultant antibody, Leung et al., J. Immunol. 154: 5919 (1995), making these carbohydrates ideal site-specific conjugation sites for drugs or chelates. However, in order to conjugate at these carbohydrates, the ribose rings must be chemically oxidized to generate reactive aldehyde groups. Aldehyde groups thus formed can be covalently bonded to the amino groups of chelates or drugs through Schiff bases. Since only the C—C bonds with hydroxyl groups attached to each carbon can be periodate-oxidized to form two aldehyde groups, the maximum number of these reactive sites is dictated by the structure and linkages of the oligosaccharide.
For example, the compositions and sequences of CH1-appended carbohydrates from two antibodies, hLL2HCN1 and hLL2HCN5, have been determined by fluorophore-assisted carbohydrate electrophoresis (FACE) 16411. Qu et al., Glycobiol. 7(6): 803-09 (1997). The structural profile of hLL2HCN1-carbohydrates revealed that about 2-4 hexose rings in an oligosaccharide chain are available for periodate oxidation. Therefore, a maximum of 8-16 aldehyde groups on average can be generated from the carbohydrate side chains of each hLL2HCN1 F(ab′)2 fragment. With the average size of hLL2HCN5-carbohydrate being 3-4 monosaccharide residues larger than that of HCN1, a higher number of maximum achievable aldehyde groups for hLL2HCN5 is expected. Under mild chemical conditions, only 1.6 and 3 molecules of DTPA were conjugated to the F(ab′)2 of hLL2HCN1 and hLL2HCN5 sites, respectively, probably due to inefficient oxidation of hexose rings under these conditions. Although harsher conditions can be used to generate more aldehyde groups, they may alter the three-dimensional structure of the antibodies and the immunoreactivities of the antibodies may suffer.
Brumeanu et al., J. Immuno. Meth. 183: 185-97 (1995), reported coupling peptides to the carbohydrate moieties of antibodies with an enzymatic procedure, in which C-6 aldehydes were generated by oxidizing the terminal galactose (Gal) residues of desialylated immunoglobulins (Igs) with galactose oxidase (GAO). Attachment of peptides is then achieved with concurrent stabilization of the Schiff bases upon mild reduction. The conjugation occurs under physiological conditions, and is specific and efficient with the average number of peptides coupled per Ig being in agreement with the estimated number of galactose equivalents. However, this method requires numerous time consuming steps and cannot be adapted for in vivo conjugation in the context of pretargeting.
There is a need, therefore, for antibodies and antibody fragments that can be conjugated at specific sites to form immunoconjugates useful in clinical applications, such as the diagnosis and treatment of cancer and infectious diseases. There also is a need for a method of making antibody and antibody fragment conjugates wherein the conjugation occurs at specific sites and does not interfere with the specific binding of the antibody or antibody fragment.